Most of our PET-CT studies to date have used 18F fluoro deoxyglucose (18F-FDG) to image the metabolism of the eukaryotic cells in TB lesions but we are also making attempts to identify the location, abundance and metabolic state of the bacteria in lesions. In an effort to identify small molecules that could be used to specifically label MTb in vivo, we capitalized on the unusually broad substrate tolerance of the MTb antigen 85 enzymes, which transfer mycolates onto structurally diverse sugars to form part of MTbs cell wall. Antigen-85 enzymes are expressed on the exterior of MTbs cell wall and incorporate exogenous trehalose (a nonmammalian disaccharide consisting of a two 1-1 &#945;, &#945; -linked glucose monomers) as either the mono- or dimycolate, even tolerating trehalose molecules containing bulky modifications. We have used this system to incorporate 18F trehalose into bacteria in the lesions of infected rabbits and 18F activity has been detected in lesions of infected rabbits by PET-CT imaging. A series of different positions and methods for attaching the 18F to the sugar are being explored to see which is most efficiently incorporated. Using trehalose should afford an improvement over the currently used 18F-FDG, as glucose is used by mammalian cells as well as bacterial, causing noise in the scans due to increased metabolism or inflammation in the host. Use of trehalose is unique to bacteria; it is not absorbed by mammalian cells, which should limit noise in PET scans. The Davis group has designed three trehalose analogs incorporating fluorine at the 2, epi-4, or 6-position of trehalose for use as PET radiotracers. The 2-fluorotrehalose synthesis is a biomemetic process, inpired by bacterial synthesis of trehalose from glucose. Chemoenzymatic synthesis of 2-fluorotrehalose (2-FTre)occurs as a one-pot cascase reaction in which hexokinase transfers a phosphate from adenosine triphosphate (ATP) to 18F-FDG (normally glucose in bacterial trehalose synthesis). OtsA then transfers the glucose from the donor UDP-glucose to the acceptor phosphorylated 18F-FDG. Dephosphorylation to give the desired product is effected by OtsB. The entire one-pot process is complete in 45 min. The advantage here is that a relatively technically facile manipulation would convert a commercially available radiotracer to a TB-specific one. We have acquired some preliminary PET-CT scan data in rabbits, one healthy, one infected with HN878 MTb. In the infected animal, four lesions were present. Two were not PET-active, and two were, although all four had similar amounts of colony forming units (CFU). Upon necropsy, the two PET-inactive lesions were extremely rigid and thick-walled, implying that uptake is related more to accessibility (i.e. vasculature) than amount of bacteria present. The t1/2 was approximately 40 min, and the radiotracer was clear from blood at 200 min post-injection, but later animals experienced unexpected toxicity within about 24 hours of the procedure. Metabolite analysis was done on a blood sample drawn 30 min post-injection, and showed that 95% of the 2-FTre was unchanged and 5% had been metabolized to 18F-FDG. In the initial procedures, the radiotracer displayed large amounts of white precipitate upon standing, contained trace protein, the pH was 11 or greater, and the saline content was approximately more than three times physiological levels, which was not reasonable for further development for use in primates and humans, so the purification strategy was reworked to produce a more physiologic final produce. We were able to use a single strong-anion exchange solid phase extraction (SAX SPE) at a pH of 8 to fully remove ADP and UDP, which lowered the saline concentration to levels nearer physiological. We then tried using a PD10 (size-exclusion column) to remove the proteins left following SAX SPE, which allowed us to remove protein to below detection limit in a Bradford assay. The final procedure gave a 75% yield (decay-corrected) over approx. 65 min synthesis time, with near-physiological pH and salinity and imaged a new set of rabbits. Lesions were again visualized, but the animals experienced unexpected toxicity within about 24 hours of the procedure. Trehalose is a non-toxic material so the cause of the toxicity had to lie elsewhere. Tests during the development of the synthesis protocol in Oxford had not shown the presence of endotoxin so other sources of toxicity were investigated, but nothing was found. Finally, we analyzed the tracer for endotoxin (or lipopolysaccharide, LPS) using a quantitative chromogenic assay (the only model currently approved by the FDA for endotoxin determination in radiotracers formulated for human use) to measure LPS levels and found extremely high levels (>6,000 EU/mL in the radiotracer). We then assayed all mixture components and found that both enzymes were contaminated with high levels (>2,000,000 EU/mL) of LPS. It was concluded that while the original high pH purification reduced the enzyme contamination, the procedure still had the original drawbacks that would prevent it from use in humans. We then turned our attention to solutions for reducing the LPS content of the radiotracer. Use of commercial Polymixin B-loaded solid phase extraction columns to purify OtsA and OtsB recovered nonfunctional enzyme. We also attempted to dialyze the enzymes against 1 M glucose to displace the endotoxin; however, the reduction in endotoxin was insufficient for further use, and repeated dialysis did not improve the endotoxin levels. Using commercially available endotoxin removal columns to purify the radiotracer post-synthesis did effectively remove LPS, but also adsorbed significant quantities of the disaccharide radiotracer and resulted in a diminished yield. Upon completion of these studies, it became increasingly obvious that the only real solution to this problem would be to express OtsA and OtsB in an organism that does not product endotoxin. We have contracted for expression and purification of the two proteins in baculovirus/insect cells and Picchi pastorii to see which system will give functional, soluble protein. The two systems have been shown to be compatible with GMP manufacturing and will allow use in humans. Neither organism produces LPS, but each requires some optimization. Moving from E. coli to baculovirus showed the higher chance of success in terms of codon usage and established methodology; however, purification typically relys on affinity tags which may affect protein function and stability. We plan to use a cleavable His-tag in order to use the native proteins in radiotracer synthesis. In P. pastorii, no affinity tags are used; however, the system is quite different than E. coli and may not give functional protein. We are evaluating both systems in parallel in order to give us the best chance of success. Both will be performed initially on a small (30 L or less) scale as a pilot, then repeated on larger scale to give GMP-quality material. Additionally, we are exploring immobilizing the proteins on solid support. Initial testing will immobilize the proteins on cyanogen bromide-activated agarose beads. We will immobilize each of the proteins separately to assay activity, then we will attempt to combine all three into a single cartridge. If successful, we could then ship these cartridges to study sites worldwide. Radiopharmacists would pass through commercial 18F-FDG and a solution of ATP and UDP-Glucose through the cartridge to produce the desired radiotracer with no specialized equipment or special training. The procedure would be operationally simple and allow TB-specific imaging to monitor course of treatment. Ideally, we would be able to rapidly assay treatment success or failure in a manner that relies only on abundance and metabolic state of the bacteria.